eGFP⁺Foxp3⁻ T cells lack suppressive activity.

<p>FACS-sorted CD4⁺eGFP⁻, CD4⁺eGFP⁺CD25^lo and CD4⁺eGFP⁺CD25^hi populations were added at varying ratios to responder T cells (Tresp) with simultaneous anti-CD3 stimulation. <i>Ex vivo</i> isolated CD4⁺CD25⁺eGFP⁺ cells (nTregs) from DEREG mice were used as control. (A) Dot plots demonstrate the purity of various FACS-sorted iTreg populations. Sorting was performed on the basis of eGFP and CD25 expression. (B) Comparison of Foxp3 expression on sorted iTreg sub-populations. Dotted line represents Foxp3 expression on live CD4⁺eGFP⁻ T cells, solid gray line represent Foxp3 expression on live CD4⁺eGFP⁺CD25^lo and solid black line represent Foxp3 expression on CD4⁺eGFP⁺CD25^hi T cells. (C) Representative histograms for dilution of proliferation dye on gated live CD4⁺ Tresp cells (left panel). Quantification of proliferated Tresp cells under various conditions (right panel). Stimulated and non-stimulated Tresp cells served as positive (Pos) and negative (Neg) controls, respectively. Error bars designate SD of triplicates from one representative of three individual experiments.</p

Culture conditions demonstrating eGFP and Foxp3 expression dichotomy amongst in vitro generated iTregs from DEREG mice.

<p>(A) Dot plots display eGFP and Foxp3 expression among the live CD4⁺ gated DEREG cells. Shown are <i>ex vivo</i> stained T cells (left), <i>in vitro</i> differentiated iTregs using soluble anti-CD3, TGF-β, RA and GM-CS- or Flt3L-derived BMDC (second and third panels respectively) and iTregs using plate-bound anti-CD3, anti-CD28, TGF-β and RA (right). (B) Differential expression of various surface antigens on eGFP⁺Foxp3⁻ (dotted line, upper panel) and eGFP⁺Foxp3⁺ (solid line, upper panel) iTregs and eGFP⁻Foxp3⁻ (dotted line, lower panel) and eGFP⁻Foxp3⁺ (solid line, lower panel) cells differentiated in the presence of TGF-β, RA, soluble anti-CD3 and DC. Gray histograms represent isotype controls. Graphs shown are representative of four individual experiments.</p

Qualitative and quantitative histological analysis of the lungs.

<p>Formalin fixed lung tissues were paraffin embedded and sections of 3 µm were cut. These sections were stained with H&E (A) or PAS (B) dyes to determine cellular infiltration and mucus production in lung, respectively. (B) Arrow heads indicate mucus producing goblet cells. (C) Overall lung extension, peri-bronchial and peri-vascular cellular infiltration and interstitial edema were considered as parameters for histological scoring in a double blinded manner. (D) Mucus secretion was assessed by measuring the surface area of mucus-containing goblet cells (Sgc) per total surface of airway epithelial area (Sep) measured. (E) The expression of the MUC5AC gene was quantified using specific primers in a quantitative PCR. Fold increase in the expression of MUC5AC in the positive control (Pos) and experimental (Pos+DT) group are plotted with respect to expression levels observed in the negative control (Neg+DT) group. Expression of GAPDH mRNA was used as internal control for data normalization. Data shown is a representative of three individual experiments using 4–10 mice in each group. Mann Whitney test was used to determine statistical significance. n.s.  =  non-significant.</p

Schematic representation of the experimental set-up.

<p>DEREG-BALB/c mice were sensitized to OVA in presence of alum on day 0 and day 14. Foxp3⁺ Treg depletion was achieved by intra-peritoneal administration of diphtheria toxin (DT) a week post the last sensitization for two consecutive days. Mice were bled through retrobulbar venous plexus one day after the last DT treatment and were analyzed for efficient depletion of Foxp3⁺ Tregs. Negative control (Neg+DT) mice were sham sensitized with PBS in alum and were also treated with DT to rule out any unspecific inflammatory effects. Positive control (Pos) mice were challenged with intra-nasal application of OVA for two consecutive days without any DT treatment. The experimental group of mice (Pos+DT) were challenged with OVA for two consecutive days 24 hr post last DT treatment. Histograms demonstrate the frequency of Foxp3⁺ cells on gated live CD4⁺ T cells, from one representative mouse of each group.</p

Th2 cytokines secretion after selective elimination of Foxp3⁺ Tregs.

<p>Lung draining mediastinal lymph node cells were pooled from each group of mice (n = 4–10) and stimulated <i>ex vivo</i> with OVA. 72 hr post stimulation cell free supernatants were collected and analyzed for Th2 cytokines (IL-4, IL-5 and IL-13) by ELISA using matched antibody pairs. Data shown are a representative of three individual experiments. Histograms represent mean values and error bars represent SD of ELISA replicates performed on four individual stimulations from each group. Mann Whitney test was used to determine statistical significance. *<i>p</i>≤0.05 and n.s. = non-significant.</p